Dark Brown Precipitate Research Articles

Characterization of Mn3 O4 -MnO2 nanocomposites biosynthesized by cell lysate of Haloferax alexandrinus GUSF-1.

Manganese oxide nanocomposites attract huge attention in various biotechnological fields due to their extensive catalytic properties. This study reports an easy, rapid, and cost-effective method of using the cell lysate of haloarchaeon, Haloferax alexandrinus GUSF-1 for the synthesis of manganese oxide nanoparticles. The reaction between the cell lysate and manganese sulfate resulted in the formation of a dark brown precipitate within 48 h at room temperature. The X-ray diffraction pattern showed the existence of Mn3 O4 and MnO2 phases consistent with the JCPDS card no. (01-075-1560 and 00-050-0866). The dark brown colloidal suspension of MnO3 -MnO2 in methanol showed maximum absorption between 220 and 260 nm. The EDX spectrum confirmed the presence of manganese and oxygen. The Transmission electron microscopy revealed the spherical morphology with an average particle size between 30 and 60 nm. The magnetic moment versus magnetic field (MH) curve, at room temperature (300 K) did not saturate even at a high magnetic field (±3T) indicating the paramagnetic nature of the prepared nanocomposite. The Atomic Emission Spectroscopic analysis showed a negligible amount of soluble manganese (0.03 ppm in 50 ppm) in the Mn3 O4 -MnO2 suspension suggesting the maximum stability of the material in the solvent over time. Interstingly, Mn3 O4 -MnO2 nanocomposites evidenced antimicrobial activity in the order of Pseudomonas aeruginosa > Salmonella typhi > Escherichia coli > Proteus vulgaris > Candida albicans > Staphylococcus aureus. Conclusively, this is the first report on the formation of Mn3 O4 -MnO2 nanocomposites using cell lysate of salt pan haloarcheon Haloferax alexandrinus GUSF-1 with antimicrobial potential.

SYNTHESIS AND PROPERTIES OF RUTHENIUM CLUSTER COMPLEXES

Methods for the synthesis of ruthenium cluster complexes based on carbonyl and amine-containing organic bifunctional ligands have been developed. The structure of the obtained combinations of clusters were determined on the basis of IR spectroscopy data, thermogravimetry, and elemental analysis. Samples of ligands I and II were obtained by condensation of carboxylic acid chlorides of cyclopentane and cyclohexane with ethylene followed by replacement of the chlorine atom by amine groups. To obtain cluster complexes of ruthenium with the synthesized ligands the ruthenium trichloride salt (RuCl3) was dissolved in water, and the calculated amount of sodium borohydride was added in portions to the resulting solution under vigorous stirring in nitrogen atmosphere. Rapidly emerging black-dispersed nanoparticles of metallic ruthenium did not precipitate. When organic ligands I and II are added, the corresponding cluster compounds III and IV are formed, which gradually over 45 min. precipitated from an aqueous solution. The resulting dark-brown precipitates were washed with distilled water and dried in a nitrogen atmosphere at a temperature of 35-400C. The melting temperatures of the synthesized compounds were determined, the components for cluster III - 2060С and cluster IV - 2220С (with decomposition). The IR spectra of cluster compounds show intense absorption bands characterizing the presence of both the ketone carbonyl group and the amine fragment. The absorption bands of ketone groups in cluster compounds are shifted towards higher frequencies compared to the initial ligands. A similar picture is also observed when comparing the IR vibrations of CN bonds in the initial ligands and the corresponding cluster compounds. The results of elemental analysis confirm the structures of cluster compounds and are in complete agreement with the concept that cluster compounds are formed upon the reduction of ruthenium salts with metal hydrides in an aqueous solution. Apparently, in this case, the most stable ruthenium clusters with tetrahedral structure are formed.. Thermogravimetric analysis made it possible to establish the presence of a peak at a temperature of 3180C with a mass number of 744.8 , corresponding to a cluster combination of four ruthenium atoms. At each stage of decomposition, the experimental mass losses are in good agreement with the calculated values. Keywords: synthesis, ruthenium, cluster complexes, amine and caryonyl containing ligands, bifunctional ligands, cyclopentyl amino ketone, cyclohexyl amino ketone.

Green fabrication of silver nanoparticles using Chloroxylon swietenia leaves and their application towards dye degradation and food borne pathogens

Green synthesized silver nanoparticles (AgNPs) are becoming an important candidate for bioremediation and biomedical applications. But in recent trends, more focus is given towards degradation of dyes and application against food pathogens. The synthesis of efficient AgNPs depends on the selection of potential biological material for synthesis. Therefore, in the present study, AgNPs were synthesized using Chloroxylon swietenia. The synthesis AgNPs was confirmed by the formation of dark brown precipitate. Further physicochemical characterization performed using XRD, FTIR, SEM and DLS showed the formation of crystalline structure, presence of functional group from the C. swietenia, dispersed spherical and rod-shaped nanoparticles (6.9 nm) and possess good stability due to the negative partial charges. The dye degrading efficacy of Chloroxylon swietenia mediated synthesized AgNPs (C-AgNPs) was >95%, 90% and >90% tested against Congo red (CR), Coomassie blue (CB) and crystal violet (CV) dye, respectively withing 24 h of treatment under optimum conditions. The antibacterial activity of C-AgNPs (10 mg/mL) was analysed against Staphylococcus nepalensis (3.03 ± 0.35 cm), Staphylococcus gallinarum (2.96 ± 0.15 cm), Bacillus subtilis (2.86 ± 0.23 cm), Enterococcous faecalis (2.8 ± 0.30 cm) and Pseudomonas stuteria (2.06 ± 0.25 cm) using Disc diffusion method, Minimum inhibitory concentration (MIC) and Minimum bactericidal activity (MBC). Therefore, the present study is the first and foremost report on C-AgNPs application as dye degrading and antibacterial agents against food dyes and pathogens. This will provide a major strategy to unveil the complications in food and packaging industries worldwide.

SARS‐CoV‐2 Viral Budding and Entry can be Modeled Using BSL‐2 Level Virus‐Like Particles

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) was first discovered in December 2019 in Wuhan, China and expeditiously spread across the globe causing a global pandemic. Research on SARS‐CoV‐2, as well as the closely related SARS‐CoV‐1 and MERS coronaviruses is restricted to BSL‐3 facilities. Such BSL‐3 classification makes SARS‐CoV‐2 research inaccessible to the majority of functioning research laboratories in the US; this becomes problematic when the collective scientific effort needs to be focused on such in the face of a pandemic. However, a minimal system capable of recapitulating different steps of the viral life cycle without using the virus' genetic material could increase accessibility. In this work, we assessed the four structural proteins from SARS‐CoV‐2 for their ability to form virus‐like particles (VLPs) from human cells to form a competent system for BSL‐2 studies of SARS‐CoV‐2. After establishing the minimal system requirements for VLP production, we examined their morphological relevance with transmission electron microscopy (TEM). VLPs produced with all four viral structural proteins were approximately 100 nm in diameter and bared the characteristic coronavirus crown or ‘corona’. We next sought to evaluate the entry competency of our VLPs. GFP‐tagged VLPs were generated by fluorescently tagging one of the four structural proteins used to produce VLPs. Once incorporation of the GFP‐tagged protein into VLPs was confirmed, we used these GFP‐VLPs to infect HEK293 cells. GFP‐VLPs indeed did enter HEK293 cells and properly colocalized with endocytic markers Rab5 and LAMP1 in accordance with live virus data. To further evaluate viral entry, we made the VLP entry assay accessible to TEM analysis by replacing the GFP tag with an ascorbate peroxidase (APEX2) tag, which when oxidized produces a dark brown precipitate visible on micrographs. APEX2‐VLPs were found to be entry‐competent as well. In addition to entry, APEX2‐VLPs yield the ability to visualize VLP assembly at the ER‐Golgi intermediary complex (ERGIC) and for the first time we show localization of the structural proteins during SARS‐CoV‐2 VLP assembly, budding, and egress. In total, this research provides ample resources for other BSL‐2 laboratories interested in joining the growing field to try and understand SARS‐CoV‐2 assembly, budding, and entry dynamics, biochemical and biophysical questions on the four structural proteins, and drug screening of viral assembly, budding, and/or entry inhibitors.

Bio-removal of phenol by the immobilized laccase on the fabricated parent and hierarchical NaY and ZSM-5 zeolites

This study was performed to synthesize and characterize NaY and ZSM-5 zeolites and their hierarchical forms (HR-Y and HR-Z) through dealumination and soft templating procedure. The Brunauer-Emmett-Teller (BET) analysis showed significant increase in the average pore diameter (DP) size for HR-Y (2.43 nm) and HR-Z (5.07 nm) compared to those of the parent types (NaY and ZSM-5: 1.85 and 2.01 nm, respectively). The mesoporous pore volumes (Vmeso) exhibited notable enhancement for HR-Y (114%) and HR-Z (566%) compared to their parent forms. Laccase (Lac) was subsequently immobilized on NaY and ZSM-5 zeolites, and their hierarchical forms by immobilization efficiency of 74.4 ± 1.4%, 71.6 ± 1.0%, 98±2.9%, and 94±1.8% for Lac@NaY, Lac@ZSM-5, Lac@HR-Y, and Lac@HR-Z, respectively. Thermal and pH stability of the immobilized laccases were enhanced compared with the free enzyme. The relative activity for the immobilized laccases was kept over 50% of its original activity after about 3 weeks of storage at 4 °C. Moreover, reusability of Lac@HR-Z and Lac@HR-Y was considerably higher (68% and 92%, respectively) than those of Lac@ZSM-5 and Lac@NaY (6% and 5.6%, respectively) after 10 cycles of reuse. Lac@HR-Z and Lac@HR-Y exhibited higher efficiency (80.5% and 89.3%, respectively) in phenol bio-removal compared with those of ZSM-5 (67.5%) and NaY (76.5%). Besides, 90.4 ± 1.3% of phenol was practically removed under optimal conditions including 12 U/mg of Lac@HR-Y, 2.6 mM of phenol, and 70 min of the reaction time. Lac@HR-Y also exhibited 56% efficiency after 10 cycles of phenol removal. Furthermore, three phenolic compounds were detected by GC-mass analysis due to phenol bio-removal in the optimal conditions after removing the dark brown precipitate of polymerized metabolites. Therefore, Lac@HR-Y could be suggested for the elimination of phenol-containing pollutants that merits further investigations.

Exercise‐mediated Wall Shear Stress Attenuates NLRP3 Inflammasome‐induced Endothelial Dysfunction in Obesity

PURPOSENLRP3 inflammasome plays a pivotal role in obesity‐induced vascular pathology through caspase‐1‐mediated IL‐1β processing, while precise mechanism by which exercise ameliorates the inflammasome remains unknown. Thus, we investigated the effect of exercise on endothelial dysfunction in obesity via NLRP3 inflammasome‐dependent mechanisms.METHODSC57BL/6J male mice were provided a control low‐fat diet (LFD; 4.5% calories from fat) or a high‐fat diet (HFD; 45% calories from fat) with a running wheel access (LF‐RUN or HF‐RUN) or a sedentary no‐wheel condition (LF‐SED or HF‐SED) for 12 to 14 weeks. Endothelium‐dependent vasodilation in response to increasing acetylcholine (ACh, 1 nmol/L to 10 μmol/L) was assessed using intact mesentery arteries isolated from mice. Mouse microvascular endothelial cell (MVEC) line EOMA was treated with palmitate (PA, 20 μmol/L) and laminar shear stress (LSS, 20 dynes/cm2): Static control (STT), PA, LSS, and LSS+PA. The NLRP3 inflammasome axis, including NLRP3, ASC, caspase‐1, p20, and IL‐1β in mice aorta and MVECs was examined by RT‐qPCR, FLICA and DAB staining and western blot.RESULTSHFD significantly increased obesity‐associated parameters while these were attenuated by running exercise. Obesity greatly decreased ACh‐induced endothelium‐dependent vasodilation compared to LF‐SED. Exercise improved reduced vasodilator response to ACh in obese mice. Incubation of caspase‐1 inhibitor (10 μmol/L) to the isolated vessel restored obesity‐induced impairment of vasodilator response similar to one of LF‐SED, LF‐RUN, and HF‐RUN. Exercise‐induced decrease of caspase‐1 and IL‐1β in endothelial cells of obese mice aorta was determined by immunohistochemistry using FLICA and DAB, respectively. Likewise, in in vitro experiment, LSS diminished inflammasome components in PA‐treated MVECs.CONCLUSIONThese findings suggest that exercise attenuates against obesity‐induced endothelial dysfunction through a shear stress‐dependent mechanism. Our finding suggests that exercise may be a therapeutic strategy for inflammation‐related vascular dysfunction in obesity.Exercise attenuates IL‐1β in endothelium of obese mice aorta.Immunohistochemical analysis of IL‐1β expression (dark brown precipitate) in the endothelial layer of the aorta. Summarized data showing area percentage of the endothelium positive for IL‐1β in aorta (n=4–6). Values are means ± SEM. * P<.05 vs. LFSED; # P<.05 vs. HF‐SED.Figure 1Inhibition of caspase‐1 abolishes endothelial dysfunction in mesentery arteries of obese mice.Incubation of the caspase‐1 inhibitor, Ac‐YVAD‐cmk, in isolated mesentery arteries from obese mice showing improved ACh‐induced endothelium‐dependent vasodilation in HF‐SED + Ac‐YVAD‐cmk (n = 3) similar to the levels of LF‐SED (n=8), HF‐RUN (n = 9) and HF‐RUN + Ac‐YVAD‐cmk (n = 7). Values are means ± SEM. ## P<.01 vs. HF‐SED.Figure 2

Assessment of long distance chasing photometer (NAG-ADF-300-2) by estimating the drug atenolol with ammonium molybdate via continuous flow injection analysis

Atenolol was used with ammonium molybdate to prove the efficiency, reliability and repeatability of the long distance chasing photometer (NAG-ADF-300-2) using continuous flow injection analysis. The method is based on reaction between atenolol and ammonium molybdate in an aqueous medium to obtain a dark brown precipitate. Optimum parameters was studied to increase the sensitivity for developed method. A linear range for calibration graph was 0.1-3.5 mmol/L for cell A and 0.3-3.5 mmol/L for cell B, and LOD 133.1680 ng/100 µL and 532.6720 ng/100 µL for cell A and cell B respectively with correlation coefficient (r) 0.9910 for cell A and 0.9901 for cell B, RSD% was lower than 1%, (n=8) for the determination of atenolol at concentration (0.5, 0.7 and 5) mmol/L respectively. The results were compared with classical method UV-Spectrophotometric at λ max=270 nm using the standard addition method via the use of t-test, at 95% confidence level. The comparison of data explain that long distance chasing photometer (NAG-ADF-300-2) is the choice with excellent extended detection and wide application.

New [Bi6O4(OH)4](CH3NHC6H4SO3)6 complex: synthesis and thermal decomposition

New compound prepared via the interaction of Bi2O3 with an aqueous solution of 4-N-methylaminobenzenesulfonic acid at 95 °C is reported. The product represents the following composition: [Bi6O4(OH)4](CH3NHC6H4SO3)6. The dark-brown precipitate derived is characterized by various methods: elemental analysis, FTIR, ICP-OES, DTA-TG and powder X-ray diffraction. Regarding TG and DTA experimental results, thermal decomposition products of new compound at three characteristic temperatures are described.

Synthesis, characterization and antimicrobial activity of Mn(ii) and Zn(ii) complexes with leucine and alanine

Background: Transition metals are essential metallic element and they exhibit great biological activity. In recent time, researches have shown significant progress in the utilization of transition metal complexes as drugs for treatment of several human disorders like cancer, infections and neurological disorders, amongst others.Objective: The study attempted to obtain neutral complexes of MLn.nH2O type (M = Mn/Zn and L = leucine/alanine), characterize, and then study their biological activities.Methods: 2mmols of the ligands were dissolved each, in 20 ml distilled water and 0.33 ml of 30% NaOH was added. Then 1 mmol of the metal salts was dissolved in 2 ml of water and stirred, the salt solution was added to the solution of the ligand and stirred. The dark brown and white precipitates for manganese amino acid complex and zinc amino acid complexe respectively were filtered off, washed, dried in air and then weighed to establish the percent of complexation. The complexes were then subjected to both spectroscopic and non-spectroscopic method of analysis.Result: The result suggested the coordination of the ligand to the central metal.Conclusion: The results suggested monoclinic crystal system for all the complexes formed. The antimicrobial studies showed an enhanced biological sensitivity for the complexes when compared with that of the ligands, and the structure of the newly synthesized complexes were proposed to be octahedral for Mn-amino acid and tetrahedral for the Zn-amino acid.  Â

Abstract DPOC-010: EARLY DETECTION: QUANTIFICATION OF B7-H4 VIA MODIFIED SANDWICH ELISA USING TRANSMISSION ELECTRON MICROSCOPY (TEM)

Abstract The American Cancer Society released statistics in 2015 that women with noninvasive cancers have a 92% chance of survival, but this rate plummets to 27% if the cancer is known to have spread. Ovarian cancer, which involves the malignant transformation of epithelial cells covering the ovary and abdominal cavity, is recognized for having dismal prognoses. This poor outcome has been largely attributed to the lack of an effective early detection method. Engineering an early–detection mechanism that is cost efficient for those unable to afford full medical scans, quick to provide real–time feedback of results for immediate treatment, and user–friendly for eventual integration into routine check–ups is the primary goal of this project. Researchers at the University of Pennsylvania have recently characterized a novel antigen of the B7 family as a promising biomarker in the diagnosis and treatment of ovarian cancer. In normal tissues, B7–H4 is present in low concentrations, however; its concentration escalates during the early stages of ovarian cancers; according to the Anderson Cancer Center in Houston, Texas, over 90% of ovarian cancer cases follow this trend Utilizing a monoclonal antibody pair to B7–H4, a method is developed to ascertain the presence of the antigen through TEM analysis. Similar to sandwich ELISA, each antibody binds to its respective epitope on the antigen, creating a sandwich. The capture antibody, JES6–1A12, is conjugated to an iron oxide magnetic nanoparticle via a carbohydrate moiety, and the detection antibody, JES6–5H4, is linked to a streptavidin coated nanoparticle via a spontaneous biotin–biotin reaction. A subsequent TEM scan outputs images of the sandwiched complex, allowing visual determination of the presence and quantity of B7–H4. Initial results from a bicinchoninic acid protein assay of a coupled iron oxide magnetic nanoparticle to JES6–1A12 detected an antigen concentration of 1510μg/mL in a 1.00mL sample. Five trials of the BCA protein assay were conducted, with each trial accounting for a potential confounding variable, including pipetting technique, dispersion of nanoparticles in the sample, and chance results. To further optimize the efficiency of JES6–1A12 conjugation to the iron oxide magnetic nanoparticle, an experiment tested eleven antibody concentrations, ranging from 0.015 uL to 15.301uL, with 10μL of nanoparticles. An absorbance scan was performed that showed 10μL of JES6–1A12 would maximize successful conjugation rate. A dot–blot immunoassay confirmed the spontaneous attachment of JES6–5H4 to the streptavidin–coated nanoparticle. A secondary gold conjugate indicated the presence of the antibody with a red color and, when combined with silver enhancement, a colorimetric change intensified the original reaction deposit and resulted in a dark brown precipitate. Results from a subsequent TEM scan are still underway. In conclusion, the modified sandwich ELISA test is hypothesized to detect both minimal and large antigen concentrations, providing key insight to the benchmark B7–H4 concentration that is indicative of early onset of ovarian cancer. This mechanism enables a rapid assessment of a woman's risk for developing advanced ovarian cancer based on her blood levels of B7–H4. Additionally, because a soluble form of B7–H4 is found in the bloodstream of cancer patients, the incorporation of the noninvasive method promises to be a cheap, mass producible diagnostic tool that will hopefully one day be incorporated into annual exams. Citation Format: Gitanjali Multani, Priyanka Multani, Preston B. Landon, Ratneshwar Lal. EARLY DETECTION: QUANTIFICATION OF B7-H4 VIA MODIFIED SANDWICH ELISA USING TRANSMISSION ELECTRON MICROSCOPY (TEM) [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr DPOC-010.

Probing the nature of AFEX-pretreated corn stover derived decomposition products that inhibit cellulase activity

Sequential fractionation of AFEX-pretreated corn stover extracts was carried out using ultra-centrifugation, ultra-filtration, and solid phase extraction to isolate various classes of pretreatment products to evaluate their inhibitory effect on cellulases. Ultra-centrifugation removed dark brown precipitates that caused no appreciable enzyme inhibition. Ultra-filtration of ultra-centrifuged AFEX-pretreated corn stover extractives using a 10 kDa molecular weight cutoff (MWCO) membrane removed additional high molecular weight components that accounted for 24-28% of the total observed enzyme inhibition while a 3 kDa MWCO membrane removed 60-65%, suggesting significant inhibition is caused by oligomeric materials. Solid phase extraction (SPE) of AFEX-pretreated corn stover extractives after ultra-centrifugation removed 34-43% of the inhibition; ultra-filtration with a 5 kDa membrane removed 44-56% of the inhibition and when this ultra-filtrate was subjected to SPE a total of 69-70% of the inhibition were removed. Mass spectrometry found several phenolic compounds among the hydrophobic inhibition removed by SPE adsorption.

Immobilization of selenium by biofilm of Shewanella putrefaciens with and without Fe(III)-citrate complex

To investigate the effect of biofilms on selenium migration, we examined selenite reduction by biofilms of an iron-reducing bacterium, Shewanella putrefaciens, under anaerobic conditions. The biofilms were grown under static conditions on culture cover glasses coated with poly-L-lysine. Optical microscopic observation of the biofilms after staining with 0.1% crystal violet solution revealed that the cells were surrounded by filamentous extracellular polymer substances. Exposure of the biofilms to aqueous selenite resulted in the formation of red precipitates, which were assigned to nanoparticulate elemental selenium using X-ray absorption near-edge structure analysis. Micrographic observation showed that the precipitates immobilized at the biofilms. We also examined the selenite reduction in the presence of Fe(III)-citrate complex. In this case, a dark brown precipitate formed at the biofilms. X-ray absorption near-edge structure analysis revealed that the precipitate was a mixed compound with elemental selenium and iron selenide. These findings indicate that biofilms of iron-reducing bacteria in the environment can immobilize selenium by reducing Se(IV) to Se(0), and Fe(III)-citrate complex promotes the reduction of Se(0) to Se(-II).

English

As public concerns about the safety of synthetic colorants increases, natural colors are assuming greater importance in the food and beverage industries. Anthocyanins are non poisonous abundant natural pigments. However, color deterioration during storage and processing limits their application as commercial colorants. The pigment extracts from black peanut skin are natural and safe colorants. The goal of this study was to study the effect of pH, temperature, metal ions, and ascorbic acid on the stability of pigments from the skins of black peanuts. The addition of Iron (Fe3+) rapidly generated black or dark brown precipitate, but other metal ions did not significantly affect the absorbance of anthocyanins. The ideal conditions for extraction of anthocyanins from black peanut skins were pH 2.0 and 60°C.   Key words: Black peanut, pre-extraction coloring, anthocyanin, stability, extrusion.

Detection of Hydrogen Peroxide by DAB Staining in Arabidopsis Leaves

In this protocol, the in situ detection of hydrogen peroxide (one of several reactive oxygen species) is described in mature Arabidopsis rosette leaves by staining with 3,3'-diaminobenzidine (DAB) using an adaptation of previous methods (Thordal-Christensen et al., 1997; Bindschedler et al., 2006; Daudi et al., 2012). DAB is oxidized by hydrogen peroxide in the presence of some haem-containing proteins, such as peroxidases, to generate a dark brown precipitate. This precipitate is exploited as a stain to detect the presence and distribution of hydrogen peroxide in plant cells. The protocol can be modified slightly to detect hydrogen peroxide in different types of plant tissue.

FM Dye Photoconversion for Visualizing Synaptic Vesicles by Electron Microscopy: Figure 1.

The synaptic vesicle is the essential organelle of the synapse. Many approaches for studying synaptic vesicle recycling have been devised, one of which, the styryl (FM) dye, is well suited for this purpose. The FM dyes have a unique set of properties that allows them to selectively label recycling vesicles: They reversibly stain, but do not permeate, membranes; hence they can specifically label membrane-bound organelles. Their quantum yield is drastically higher when bound to membranes than when in aqueous solution. FM dyes can also be used as endocytic markers in electron microscopy (EM) through a procedure termed photoconversion (or photooxidation), as described here. Fluorescent dye molecules generate free radicals (reactive oxygen species) when subjected to strong illumination. These short-lived radicals readily oxidize any molecules found in the immediate vicinity of the fluorophore. When photoconversion of FM dyes is performed while the preparation is bathing in diaminobenzidine (DAB), a dark brown precipitate forms after the DAB is oxidized. Thus, illumination turns FM-labeled organelles into dark electron-dense ones. The technique results in a substantial increase in the resolution of FM dye labeling studies (with the obvious caveat that it is restricted to fixed preparations).

Study of the Gold Extraction Using Tetra N-Butyl Ammonium Chloride-Chloroform

The existence of the AuCl4- ion in the solution as the function of pH before performing the extraction of gold in the system of tetra N-butylammonium chloride (TBACI) -chloroform has been studied. The experimental data showed that AuCl4- ion was hydrolyzed at pH 5-10 and, an amorf dark-brown precipitate was appeared at pH 11-14. Amount of gold in the solution at pH 14 before extraction was around 70%. Study of the extraction has been carried out by investigating the influence of pH and TBACI concentration on the extraction efficiency. The experimental result indicated that TBACI was very efficient extractant for the extraction of gold from aqueous halide with the efficiency higher than 99%. The extraction of Gold in the TBACI-chloroform was effective at pH 0-4 with minimum concentration of TBACI 10-3 M, and the calculated Kex (extraction constant) was 5.07x10-4.

Peroxidase-catalyzed copolymerization of syringaldehyde and bisphenol A

Syringaldehyde, one of the major derivatives of lignin, was copolymerized with bisphenol A via a CiP ( Coprinus cinereus peroxidase)-catalyzed reaction. Although syringaldehyde was not polymerized to a solid polymer, the copolymer with bisphenol A was obtained as a dark brown powdery precipitate. The relatively hydrophobic solvent, 2-propanol, gave a better yield (yield = 95%) than hydrophilic solvents, such as methanol, ethanol or acetone. Characteristic signals corresponding to the aldehyde group of syringaldehyde in the copolymer were detected in the FT-IR and 13C NMR spectrum. The ratio of syringaldehyde incorporated into the copolymer was estimated by measuring the amount of monomers consumed (syringaldehyde and bisphenol A), which proportionally increased up to 80 mol% on increasing the initial ratio of syringaldehyde to bisphenol A. TGA (thermogravimetric analysis) showed that the thermally crosslinked copolymer (syringaldehyde:bisphenol A = 1:1, w/w) had a much higher thermal resistance to thermal degradation than poly(bisphenol A); 36% residue still remained under a nitrogen atmosphere, even over 800 °C. This implies that the copolymer of syringaldehyde and bisphenol A could be a new thermally stable material originating from renewable resources.

The synthesis and thermal degradation products of the C–H bond activating complex [(diimine)Pt(Me)(OSO2CF3)

Design of a selective homogeneous methane functionalizing catalyst based on [(ArN=CRCR=NAr)Pt(Me)(L)]+ requires knowledge of its stability in various reaction media, particularly water. Reaction of (diimine)PtMe2 (1) (diimine = ArN=CRCR=NAr, Ar = 2, 6-Me2C6H3, R = Me) with HOTf (OTf = OSO2CF3) gives the methane activating compound (diimine)Pt(Me)OTf (3). When varying amounts of H2O are added during the synthesis of 3, competing degradation pathways lead to two different characterizable products. With only trace amounts of water, two dimeric species, [(diimine)Pt(μ-Cl)(μ-OH)Pt(diimine)](OTf)2 (6) and [(diimine)Pt(μ-OH)2Pt(diimine)](OTf)2 (7), are isolated, in addition to an uncharacterized dark brown precipitate. When an excess of H2O is added, the aquo species [(diimine)Pt(Me)(H2O)][OTf] (5) is first observed, which then reacts further to give a dark brown precipitate and 7. The structures of 1, 6, and 7 are presented. Both 6 and 7 exhibit unusual conformations for their respective classes. Compound 6 has a rarely observed planar conformation, while 7 has an unusual bifurcated H-bonding motif between the bridging OH-groups and a triflate anion, with a highly bent conformation.

Spontaneous HIV-1 replication in a B-lymphoblastoid cell line obtained from an HIV-1-positive patient with undetectable plasma viral load

Spontaneous HIV-1 replication in a B-lymphoblastoid cell line obtained from an HIV-1-positive patient with undetectable plasma viral load

A new rapid acetylcholinesterase histochemical method for the intraoperative diagnosis of Hirschsprung's disease and intestinal neuronal dysplasia.

The most commonly used acetylcholinesterase (AChE) method for the diagnosis of Hirschsprung's disease (HD) and intestinal neuronal dysplasia (IND) was first introduced in 1964 by Morris Karnovsky and Logan Roots. This technique requires about 80 - 120 minutes incubation time and cannot be used for the intraoperative diagnosis of HD and IND. To avoid these limitations, in 1994 Kobayashi et al first proposed an accelerated modified method in two different versions, the first using diaminobenzydine (DAB) reagent, the second using 4-chloro-1-naphthol as final reagent. In the present study, we propose a new rapid variation of AChE staining which avoids the use of DAB and naphthol, notably toxic reagents, but follows the same acceleration principle of Kobayashi's technique. Our modified rapid AChE requires a total incubation time of only 8 minutes, which is compatible with intraoperative histochemical examination purposes. Intraoperative seromuscular or full-thickness intestinal biopsies were obtained from 92 children affected by intestinal dysganglionoses. The biopsies were frozen and cut in 15 microm cryostatic sections. Rapid AChE was performed with a special incubation medium using 3-amino-9 ethylcarbazole (AEC) as chromogenic substance. The two complementary histochemical techniques alpha-naphthylesterase (ANE) and lactate-dehydrogenase (LDH) were also used intraoperatively for the staining of ganglion cells. The diagnosis was confirmed postoperatively with conventional AChE Karnovsky technique, comparing the extensions of hyperganglionic, hypoganglionic and aganglionic segments in each studied case. The new rapid AChE modified method can identify ganglion cells and fibers using a dark brown precipitate. In all the cases studied, the intestinal innervation pattern identified with this modified technique was similar to that obtained with Karnovsky AchE. Seventy-eight HD, 8 isolated IND and 6 HD associated with an evident IND segment were diagnosed. This new rapid AChE histochemical technique avoids the use of DAB and naphthol, and can thus be considered safe for operators. Rapid AChE is a valid tool for both the evaluation of aganglionosis extension and for the identification of IND pattern during surgery. We recommend this very reliable method for the intraoperative diagnosis of HD and IND, in association with other enzymatic markers of ganglion cells (ANE or LDH). We propose the following diagnostic protocols: a) for preoperative histochemical study: conventional AChE plus LDH and NADPH-diaphorase; b) for intraoperative study: rapid AChE plus ANE.